1. Field of the Invention
The present invention relates to a microvolume electrochemical detection assay. More specifically, the present invention relates to microscale structures having an analyte immobilizing surface and at least one electrode separate from the immobilizing surface. The microscale devices are used in conjunction with an immobilization assay that utilizes an electroactive complex to generate a current that may be detected by the electrode. The small volumes and sensitivity of the assays used in conjunction with microstructures provides an extremely rapid detection method having superior sensitivity to existing methods.
2. Prior Art
There is a great need to miniaturize the analytical methodologies and instrumentation for making rapid and sensitive analyses in biological, environmental, medical and food applications. The interest in decreasing the analysis volume is of value for samples which are precious, expensive, require high spatial chemical resolution, and need improved throughput (e.g. more sample capacity in a smaller space). This does not necessarily demand better detection limits. However, improvements in limits of detection increase sensitivity and accuracy and are also beneficial for dilute analyte in small samples.
One analytical methodology that has been successful at providing the high specificity and selectivity, and which is transferable to small volumes is the immunoassay. This approach has been primarily designed for medical diagnostics, and combines the highly specific antigen (Ag)/antibody (Ab) interaction with the sensitivity of a transducer system that may be optical, radiological, piezoelectric or electrochemical. Immunoassays with electrochemical detection are desirable for a wider range of uses because highly specific and precise current measurements can be performed with simple instrumentation, using opaque device materials, and in colored and turbid samples minimizing prior pre-treatment procedures.
Some of the smallest analyzed volumes have been reported for homogeneous immunoassays, although with poorer detection limits than those of the present invention. One type of small homogeneous assay involves laser induced fluorescence detection combined with fast separation of bound and unbound antibody-antigen complexes in microfluidic systems. Another has the advantages of the simpler electrochemical detection and can be performed in a drop (600 pL). The latter device, however, does not include a separation step, and therefore may be susceptible to interferences from other sample species. Recently, an example of an electrokinetically-driven microfluidic chip for heterogeneous bioassays using about 118 nL volume and assay times below 5 min was reported. Detection was performed with laser induced fluorescence. Complications include modifying the walls of the channel, which affects the electro osmotic flow, and elution of fluorescent-labeled-immobilized species to bring them to the detection site. Low detection limits (pg/mL or fM) were not possible.
Detection immediately adjacent to the surface-immobilized immuno components should supply the largest signals with the shortest incubation periods. This has been accomplished with scanning electrochemical microscopy (SECM). Detection limits as low as 5.25 pg/mL have been obtained. However, long total assay times were required (1 h and up). Only the sample volume is small (10's of pL36 up to several μL) which is spotted onto a surface (and dried, followed by rinsing) or immobilized onto magnetic immuno beads first, which are subsequently transferred to a surface for electrochemical detection. Yet, the enzymatic generation of electrochemically-detected species is carried out on large volumes that must keep both the SECM electrode and the auxiliary/reference electrodes in electrochemical contact. Consequently, this setup is not well-suited for integration with small volume handling or automation and has added complexity due to the SECM instrumentation and operation.
Heterogeneous immunoassays for human serum albumin on a thick-film electrochemical device have been reported. The immuno components are also immobilized adjacent to the detecting electrode and small volumes may be used at all stages of the immunoassays. However, unlike the present invention the immuno components are attached in a noncovalent fashion to all surfaces (instead of selected ones), the overall dimensions are large (several millimeters) and therefore, the volumes must be larger (30 μL) to cover electrodes and modified surfaces, the area of the immuno active surface exposed to solution is less well defined because it depends upon the size of the drop and wetting properties of the substrate, and the detection (based upon potentiometric stripping analysis) yields detection limits that are higher (0.2 μg/mL).
General immunoassay procedure involves immobilization of the primary antibody (Ab, rat-anti mouse IgG), followed by exposure to a sequence of solutions containing the antigen (Ag, mouse IgG), the secondary antibody conjugated to an enzyme label (AP-Ab, rat anti mouse IgG and alkaline phosphatase), and p-aminophenyl phosphate (PAPp). The AP converts PAPp to p-aminophenol (PAPR, the “R” is intended to distinguish the reduced form from the oxidized form, PAPo, the quinoneimine) which is electrochemically reversible at potentials that do not interfere with reduction of oxygen and water at pH 9.0, where AP exhibits optimum activity. In addition, PAPR does not cause electrode fouling, unlike phenol whose precursor, phenylphosphate, is often used as the enzyme substrate. Although PAPR undergoes air and light oxidation, these are easily prevented on small scales and short time frames. Picomole detection limits for PAPR and femtogram detection limits for IgG achieved in microelectrochemical immunoassays using PAPp volumes ranging from 20 μl to 360 μL have been reported previously. In capillary immunoassays with electrochemical detection, the lowest detection limit reported thus far is 3000 molecules of mouse IgG using a volume of 70 μL and a 30 min or assay time. Those skilled in the art will recognize the above described assay as a sandwich-type immunoassay and will appreciate that this is only one of many immunoassays. Alternatives include competitive binding immunoassays and immunoassays utilizing a more general physisabsorbing material other than a primary antibody.
Immunoassays are only one category of a very wide variety of surface immobilization chemical detection assays. Northern and southern blot assays are well known techniques for detecting specific polynucleotide sequences. They involve surface immobilization of polynucleotides. Surfaces having one or more lipid layers may be used to immobilize and detect compounds having hydrophobic regions. Molecular interactions may also be taken advantage of to develop surface immobilization chemical detection assays. When two molecules are known to bind to one another, one may be covalently attached to a substrate. The substrate is then exposed to a sample such that the other interacting molecule is given an opportunity to bind to the substrate bound molecule. The substrate is then rinsed leaving only bound analyte on the substrate. A number of detecting methods may then be applied to the surface. Detecting methods include using secondary antibodies as described above, detecting the bi-products of an enzymatic reaction characteristic of the analyte, spectroscopy, flourescent, electrochemical analysis or other methods known to those skilled in the art.
These assays generally require a laboratory setting. A person wishing to analyze a sample with one of the above described assays most usually sends the sample to a laboratory. Even while in a laboratory, many chemical detection assays take a relatively long period of time.
The disadvantages of immunofluorescence assays (IFA) include their low recovery efficiency, long processing times, the need for highly trained analysts and high cost. In addition, IFA detection often involves the time consuming and skill intensive step of looking at water sludge under a microscope for analytes that have been labeled with a fluorescent antibody. It is also often difficult to distinguish oocysts from debris bound non-specifically by the antibodies. The procedure is expensive and often takes days to complete.
Flow cytometry is a method used to detect parasitic contamination of water samples. Flow cytometry techniques can quantify microorganisms but involves much preparation, and time and require extremely expensive equipment.
Numerous problems are associated with prior art methods of detecting microorganisms and biological molecules in water and environmental samples. In addition to those mentioned and the general lack of precise, recitable assays, prior art techniques generally require that samples be transferred to a laboratory or to another remote location for the conduct of the assay. Prior art techniques lack the requisite reliability, speed and sensitivity to accurately detect microorganisms and biological compounds in contaminated water samples.
It is crucial that specific, rapid and highly sensitive assays be developed to detect bacteria and toxins accurately and reliably. The known methods of enzyme immunoassays and immunofluorescence do not fulfill these requirements. The source, viability and pathogenicity of oocysts found in water or other environmental samples cannot be reliably determined using prior art methods. There is a need for routine epidemiological surveillance and environmental monitoring that can be conducted on site to provide early detection of the parasite.
It is therefore desirable to provide a method for rapid chemical detection.
It is also desirable to provide a highly sensitive method for detecting low amounts of analyte in a very small amount of sample.
It is also desirable to provide a method for detecting an analyte in a small sample having very high accuracy.